牛分枝杆菌融合基因hsp65-esat6的原核表达及产物的纯化

以牛分枝杆菌ValleeⅢ株基因组DNA为模板扩增hsp65和esat6基因。采用重叠延伸剪接技术（SOE）获得了融合基因hsp65-esat6，将hsp65-esat6连接到原核表达载体pET32a（＋）上，构建重组质粒pET65-E6，将其转化到大肠杆菌BL21（DE3）感受态细胞中，以IPTG诱导（终浓度为1mmol／L），表达产物进行SDS—PAGE分析。以Ni^2＋亲和层析柱纯化表达的融合蛋白和Western—blot分析该融合蛋白的免疫活性。结果表明：Hsp65-ESAT6融合蛋白以包涵体形式表达。表达的融合蛋白裂解为两部分，即58ku和30ku，二者相加与预测大小88ku相符。纯化的融合蛋白能与抗牛分枝杆菌阳性血清发生反应。     

牛整合素α4亚基片段的原核表达及抗体制备

参照GenBank中牛整合素α4基因序列,合成了牛整合素α4亚基753bp（192～944nt）的cDNA片段,克隆至原核表达载体pET-28a（＋）中,转化入BL21,IPTG诱导表达。表达产物经Western blot鉴定,结果表明成功表达了相对分子质量约为30 000的α4蛋白。超声破碎细菌后,目的蛋白主要以包涵体形式存在。蛋白纯化后进一步复性,免疫大鼠获得多抗,间接ELISA检测多克隆抗体效价为1∶32 000。这为进一步研究牛整合素α4β7的功能奠定了基础。     

羊白介素12受体β2链的表达与纯化

利用PCR和基因合成获得了野生型羊IL-12Rβ2和密码子优化的IL-12Rβ2-A基因,亚克隆至pET30a(+),SDS-PAGE对这2个重组载体的表达结果进行比较,并优化了pET30a-12Rβ2-A的最佳表达条件;通过Western blot对镍琼脂糖凝胶纯化的融合蛋白His-12Rβ2进行了检测。结果显示,pET30a-12Rβ2-A的表达量明显高于pET30a-12Rβ2的表达;在筛选的pET30a-12Rβ2-A最佳表达和纯化条件下,成功获得了高纯度的融合蛋白His-12Rβ2,达到了预期的目标,为后续的功能研究奠定了基础。     

猪FcRn胞浆尾区的克隆表达及多克隆抗体制备

采用RT-PCR方法从猪肝脏总RNA中克隆猪FcRn基因,然后利用PCR技术亚克隆猪FcRn胞浆尾区（FcRn-CT）,构建重组原核表达质粒KG-CT和pET32a-CT,分别在大肠杆菌BL21中获得了高效表达,并利用Glutathione Sepharose 4B和HisTrap FF crude亲和层析柱纯化目的蛋白。用纯化蛋白KG-CT免疫新西兰白兔,制备了多克隆抗体。利用纯化蛋白pET32a-CT建立了间接ELISA法,检测多抗效价达1∶32 000。Western blot检测结果表明该多抗特异性良好,为进一步研究FcRn在猪体内的表达分布及功能研究奠定了基础。     

猪圆环病毒2型ORF2基因的原核表达及其免疫原性检测

以病猪脾脏组织为材料,提取总RNA,通过RT-PCR得到579 bp的猪圆环病毒2型(PCV-2)/ORF2片段,与原核表达载体pET32a重组,通过菌落PCR鉴定、双酶切鉴定及测序鉴定后,证实重组质粒pET32a/ORF2构建成功,将其转化入表达菌Balgold,通过IPTG诱导表达,利用Ni-NTA亲和层析纯化目的产物,Western blot验证其免疫原性,重组蛋白免疫Balb/c小鼠,检测小鼠体内中和抗体滴度。获得的重组蛋白包涵体的形式出现,表达量占菌体总蛋白的30%,变性纯化后纯度达90%以上,Western blot证实重组蛋白能与PCV-2阳性血清发生反应,免疫Balb/c小鼠45 d后中和抗体滴度达到1∶22。     

猪圆环病毒2型多抗原表位串联在大肠杆菌中表达与反应原性分析

为将猪圆环病毒2型(PCV2)的多个抗原表位串联,并在大肠杆菌中表达,本研究人工合成PCV2多抗原表位串联的编码序列并与pET32a(+)载体连接构建重组表达质粒。将其转化到BL21感受态细胞,经IPTG诱导表达。检测结果显示,多抗原表位串联基因在大肠杆菌中获得表达并以包涵体形式存在;表达的融合蛋白分子量约为27 ku。将包涵体溶解并经Ni-His柱纯化得到纯化的目的蛋白,western blot和ELISA结果显示,该表达产物具有较好的反应原性。     

堆形艾美耳球虫巨噬细胞移动抑制因子的克隆和表达

本研究扩增和克隆了堆形艾美耳球虫巨噬细胞移动抑制因子,并在大肠杆菌中表达出MIF重组蛋白。根据GenBank中的MIF mRNA序列,设计特异引物,采用PCR方法以堆形艾美耳球虫孢子化卵囊cDNA文库为模板扩增获得MIF基因,将MIF基因片段连接到原核表达载体pET-28a（＋）,构建重组表达质粒pET28a—MIF,并在大肠杆菌BL21（DE3）中诱导表达,对表达产物进行Western blot分析。结果从堆形艾美耳球虫孢子化卵囊cDNA文库中扩增出MIF基因片段,长度为709bp;经序列分析,扩增片段的核苷酸序列与GenBank中登录的MIF序列的同源性为99．5％;经SDS-PAGE检测表明重组质粒pET（28a）-MIF在大肠杆菌中以包涵体形式表达;Western blot分析证明堆形艾美耳球虫抗血清可与重组表达蛋白特异性结合。本研究结果为进一步研究该基因的功能奠定了基础。     

隆林黑猪氨肽酶N基因的生物信息学分析和原核表达

本研究旨在进行猪氨肽酶N（porcine aminopeptidase N,pAPN）基因扩增和生物信息学分析,并在大肠杆菌中进行表达。从三元杂商品猪和广西地方猪隆林黑猪中扩增出pAPN基因,通过ExPASy和Mega 6.0等软件对其进行生物信息学和系统进化分析;将pAPN克隆到原核表达载体pET-32a（+）,构建重组质粒pET-32a-pAPN,使其在大肠杆菌Rosetta（DE3）感受态细胞中表达。经IPTG诱导表达、纯化,产物用SDS-PAGE和Western blotting进行分析。结果显示,隆林黑猪和三元杂商品猪的pAPN基因长2 949 bp,开放性阅读框为2 892 bp,编码963个氨基酸,同源性为100%,与GenBank数据库中公布的标准序列（登录号：NM_214277）相比,隆林黑猪共有5处氨基酸突变,其中Phe82Asn、Leu107Phe、Leu108Ile和Ser330Pro 4处突变位于pAPN酶催化活性区域,Val621Ile突变位于APN病毒结合区域。pAPN为不稳定的亲水性蛋白,跨膜区位于第17~33位氨基酸,有12个N-糖基化位点,33个B细胞抗原表位预测位点,三级结构为同源二聚体。Val621Ile突变可能影响APN结合病毒的能力。试验成功构建重组质粒pET-32a-pAPN,SDS-PAGE鉴定以包涵体形式表达,纯化后的蛋白在123 ku处有明显条带,且具有良好的抗原性,为进一步获得多克隆抗体、研究该基因和TGEV、PEDV、PDCoV的关系奠定基础。     

牛分枝杆菌Ag85A基因在大肠埃希氏菌中的表达

以BamHⅠ和EcoRⅠ双酶切已构建的pGEM-T-85A和pET28a（＋），并将纯化的Ag85A基因亚克隆至pET28a（＋）中，构建出原核表达质粒pET28a-85A。将pET28a-85A转化至感受态E．coli BL21（DE3）中，经IPTG诱导和SDS-PAGE分析，可见约32ku的外源蛋白带。Western-blotting分析表明，该蛋白具有牛分枝杆菌的抗原性。     

隆林黑猪氨肽酶N基因的生物信息学分析和原核表达

本研究旨在进行猪氨肽酶N(porcine aminopeptidase N,pAPN)基因扩增和生物信息学分析,并在大肠杆菌中进行表达。从三元杂商品猪和广西地方猪隆林黑猪中扩增出pAPN基因,通过ExPASy和Mega 6.0等软件对其进行生物信息学和系统进化分析;将pAPN克隆到原核表达载体pET-32a(+),构建重组质粒pET-32a-pAPN,使其在大肠杆菌Rosetta(DE3)感受态细胞中表达。经IPTG诱导表达、纯化,产物用SDS-PAGE和Western blotting进行分析。结果显示,隆林黑猪和三元杂商品猪的pAPN基因长2 949 bp,开放性阅读框为2 892 bp,编码963个氨基酸,同源性为100%,与GenBank数据库中公布的标准序列(登录号:NM_214277)相比,隆林黑猪共有5处氨基酸突变,其中Phe82Asn、Leu107Phe、Leu108Ile和Ser330Pro 4处突变位于pAPN酶催化活性区域,Val621Ile突变位于APN病毒结合区域。pAPN为不稳定的亲水性蛋白,跨膜区位于第17～33位氨基酸,有12个N-糖基化位点,33个B细胞抗原表位预测位点,三级结构为同源二聚体。Val621Ile突变可能影响APN结合病毒的能力。试验成功构建重组质粒pET-32a-pAPN,SDS-PAGE鉴定以包涵体形式表达,纯化后的蛋白在123 ku处有明显条带,且具有良好的抗原性,为进一步获得多克隆抗体、研究该基因和TGEV、PEDV、PDCoV的关系奠定基础。     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

乳酸杆菌作为益生菌广泛用于人和动物。本文综述了乳酸杆菌改善宿主健康的机制。乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道。文章重点讨论了乳酸杆菌表面成分（表面蛋白、脂磷壁酸和肽聚糖）与肠道受体（Ｃ型凝集素受体、Ｔｏｌｌ样受体和 Ｎｏｄ样受体）,阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制。     

中华人民共和国农业部公告 第267号

为贯彻落实《兽药生产质量管理规范》(简称《兽药GMP》),进一步推动兽药GMP实施进程,我部制定了《兽药生产质量管理规范检查验收办法》,现予公告。本公告自2003年6月1日起施行。附件:兽药生产质量管理规范检查验收办法二○○三年四月十日第一章 总则 第一条 为推动《兽药生产质量管理规范》(以下简称兽药GMP)的实施,规范兽药GMP检查验收工作,制定本办法。 第二条 农业部负责全国兽药GMP管理和检查验收工作;负责制修订兽药GMP检查验收管理规定;负责兽药GMP检查员队伍建设和监督管理工作,负责国际兽药贸易中GMP互认工作。 …     

鸡败血支原体垂直传播模式及其阻断方法

以国际标准强毒Ｒ株人工感染非免疫产蛋鸡,定时扑杀,分别从鼻窦、眶下孔、气管、肺、气囊、卵巢和输卵管分离ＭＧ,并收集感染鸡所产蛋分离ＭＧ。结果表明,人工感染４８小时后上、下呼吸道及肺已被全面感染,９６小时气囊已被感染,１２０小时输卵管已能分离到ＭＧ,卵巢始终分离不到ＭＧ。人工感染鸡自１４４小时便能在其所产蛋中分离出ＭＧ。药物治疗能在７２小时内消除感染,油乳剂苗则需２４天后逐渐降低蛋内ＭＧ分离率,药物卵内注射、种蛋药浴、高温处理均能杀死卵内ＭＧ,但以研制的种蛋浸泡剂药浴效果为最好。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.